Undiagnosed Diseases Program, Office of Rare Diseases Research, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland;Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland; and

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland;Undiagnosed Diseases Program, Office of Rare Diseases Research, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland;Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland; and

Summary

Background and objectives Elevated serum vitamin D with hypercalciuria can result in nephrocalcinosis and nephrolithiasis. This study evaluated the cause of excess 1,25-dihydroxycholecalciferol (1α,25(OH)2D3) in the development of those disorders in two individuals.

Design, setting, participants, & measurements Two patients with elevated vitamin D levels and nephrocalcinosis or nephrolithiasis were investigated at the National Institutes of Health (NIH) Clinical Center and the NIH Undiagnosed Diseases Program, by measuring calcium, phosphate, and vitamin D metabolites, and by performing CYP24A1 mutation analysis.

Results Both patients exhibited hypercalciuria, hypercalcemia, low parathyroid hormone, elevated vitamin D (1α,25(OH)2D3), normal 25-OHD3, decreased 24,25(OH)2D, and undetectable activity of 1,25(OH)2D-24-hydroxylase (CYP24A1), the enzyme that inactivates 1α,25(OH)2D3. Both patients had bi-allelic mutations in CYP24A1 leading to loss of function of this enzyme. On the basis of dbSNP data, the frequency of predicted deleterious bi-allelic CYP24A1 variants in the general population is estimated to be as high as 4%–20%.

Conclusions The results of this study show that 1,25(OH)2D-24-hydroxylase deficiency due to bi-allelic mutations in CYP24A1 causes elevated serum vitamin D, hypercalciuria, nephrocalcinosis, and renal stones.

Introduction

Although nephrocalcinosis and nephrolithiasis are distinct entities, evidence suggests a common underlying mechanism (1). Nephrocalcinosis refers to the diffuse precipitation of calcium salts within the tubules, tubular epithelium, and/or interstitial tissue of the kidney (2). It involves the medulla in 98% of cases, and is readily detected by ultrasonography or computed tomography (CT). Nephrolithiasis refers to renal stones, generally visible on plain radiographs, ultrasounds, or CT scans; the majority of renal stones are composed of calcium salts (3). Because calcium precipitation is common to both nephrocalcinosis and nephrolithiasis, the two entities often occur together (4). In addition, both nephrolithiasis and nephrocalcinosis frequently result from metabolic abnormalities such as hyperphosphaturia, hyperoxaluria, hypocitruria, hyperuricosuria, defective urinary acidification, and especially hypercalciuria (5). Approximately 50% of patients with nephrolithiasis (6) and 14%–27% of non-stone formers (7) have hypercalciuria.

Previous classifications of hypercalciuria have been revised in view of recent evidence of intrinsic alterations of calcium metabolism associated with this phenomenon. Calcium metabolism is regulated by parathyroid hormone (PTH), calcitonin, and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), the active form of vitamin D. Today, hypercalciuria is considered a complex trait, governed by several genetic mechanisms.

One of those mechanisms involves 1α,25(OH)2D3, which acts on calcium and phosphate to stimulate mobilization from bone, reabsorption by the kidney, and absorption by the intestine, 1α,25(OH)2D3 is formed through 1-hydroxylation of 25(OH)D3 (cholecalciferol) by the gene product of CYP27B1, which is present in many tissues; 25(OH)D3 is formed by 25-hydroxylation of vitamin D in the liver (Figure 1) (8). Excessive 1α,25(OH)2D3 leads to absorptive hypercalcemia and/or hypercalciuria (9), as first described in 1928 by Kreitmair and Moll (10).

Vitamin D metabolism. The first step of activation, 25-hydroxylation by CYP2R1 and CYP27A1, occurs in the liver. The second step, 1α-hydroxylation by CYP27B1 to yield active vitamin D (i.e., 1α,25(OH)2D3), occurs in the kidney. Inactivation of vitamin D occurs via the C-23 and C-24 oxidation pathways, catalyzed by CYP24A1 in the kidney. Calcitroic acid has no biologic activity. Larger type emphasizes important metabolites.

Both major vitamin D metabolites, 25(OH)D3 and 1α,25(OH)2D3, are inactivated by 24-hydroxylation, a process catalyzed by 1,25(OH)2D-24-hydroxylase (CYP24A1), a mitochondrial cytochrome P-450 mixed-function oxidase present largely in intestine and kidney. Bi-allelic mutations in CYP24A1 and reduced activity of the enzyme have been associated with elevated levels of 1α,25(OH)2D3 in individuals given large amounts of vitamin 25(OH)D3 (11). In addition, a recent report demonstrated reduced 1,25(OH)2D-24-hydroxylase activity in patients with monoallelic CYP24A1 mutations (12).

We present a young boy with nephrocalcinosis and an adult male patient with nephrolithiasis, both of whom have molecular and biochemical defects in CYP24A1, leading to elevated levels of 1α,25(OH)2D3 and hypercalciuria.

Materials and Methods

Patients

Patients were enrolled in clinical protocol 76-HG-0238, “Diagnosis and Treatment of Patients with Inborn Errors of Metabolism and Other Genetic Disorders” and were evaluated at the National Institutes of Health (NIH) Clinical Center. Written, informed consent was obtained. Patient 2 was enrolled in the NIH Undiagnosed Diseases Program (UDP) (13,14), dedicated to investigating the basic defects underlying undiagnosed disorders.

Biochemical Studies

Serum 24,25-(OH)2D levels were measured by RIA after lipid extraction and purification by high-performance liquid chromatography (HPLC). The intra-assay and interassay coefficients of variation (CVs) were 9.3% and 11.8% , respectively (Heartland Assays LLC) (17). 1,25(OH)2 vitamin D testing was performed by immunoextraction and liquid chromatography–tandem mass spectrometry, similar to published methods (18). 1,25(OH)2D2 interassay imprecision was 8.5% and 8% at levels of 67 and 219 pg/ml, respectively. Intact human fibroblast growth factor-23 (FGF-23) was measured using a commercially available ELISA kit (Kainos, Tokyo, Japan). The CV for the FGF-23 assay was <5%, as reported by the manufacturer.

Results

Patients

Patient 1 (family I, patient I-2.2) is a 9-year-old boy with nephrocalcinosis diagnosed by routine renal ultrasonography performed because of a urinary tract infection at 3 years of age. Initial investigations outside the NIH showed hypercalciuria up to 8 mg/kg per day (normal <4.0 mg/kg per day) and increased plasma 1α,25(OH)2D3 to 86 pg/ml (normal 18–64 pg/ml). Repeated 24-hour urine evaluations revealed significant hypercalciuria, and no hyperuricosuria or oxaluria (Table 1), elevated serum calcium levels and appropriately suppressed intact PTH, as well as normal serum 25(OH)D2, elevated 1α,25(OH)2D3, and decreased 24,25-(OH)2D (Table 2). Renal ultrasonography performed at the NIH showed extensive medullary nephrocalcinosis. A dual energy x-ray absorptiometry scan yielded Z-scores of −1.4 for the spine, 0.2 for the femoral neck, and −1.3 for the forearm. The patient’s unaffected dizygotic twin brother did not have kidney stones or nephrocalcinosis, and neither did any other family member.

Patient 2 (family II, patient II-2.2), a 38 year-old man, had recurrent kidney stones since age 25 years, hypercalcemia, hypercalciuria, and marked elevation of 1α,25(OH)2D3. His 24-hour urine evaluations revealed hypercalciuria without hyperuricosuria or oxaluria (Table 1), high serum calcium levels and suppressed intact PTH, normal serum 25(OH)D3, high 1α,25(OH)2D3, and low 24,25-(OH)2D (Table 2). He had an elevated fractional excretion of phosphate (Table 1). An abdominal CT scan performed at age 34 years demonstrated a large renal calculus (25×34 mm) in the left kidney. Treatments included extracorporeal shockwave lithotripsy, percutaneous nephrolithotomy with subsequent residual stone fragments, Harrington rod placement for scoliosis, thiazide diuretics, sodium cellulose phosphate, and, after his admission to the NIH Clinical Center, ketoconazole. Stone analysis of patient 2, performed at the Mayo Clinic, demonstrated 100% calcium phosphate (brushite). A gallium scan showed no evidence of granulomatous disease. The dual energy x-ray absorptiometry Z-scores and T-scores were −2.8 and −2.8 in the spine, −1.3 and −1.8 in the femoral neck, and −2.8 and −3.0 in the forearm, respectively,. The plasma FGF-23 level, a possible contributing factor to the patient’s hyperphosphaturia, was 64 RU/ml, within the normal range (63±45 SD). The patient had decreased urine calcium after administration of sodium cellulose phosphate, confirming the presence of increased enteric calcium absorption (data not shown). On ketoconazole therapy (800 mg per day in divided doses), the patient’s serum 1,25(OH)2D3, serum calcium, and urine calcium decreased, and the PTH returned to normal (Figure 2). His male sibling (II-2.1) had a questionable history of renal stones.

Effect of ketoconazole treatment in patient 2 on 1,25 vitamin D levels, hypercalcemia, hypercalciuria, and parathyroid hormone. Patient 2 was treated with ketoconazole and the dosage was escalated to 800 mg per day in divided doses. The effects monitored before the initiation of therapy, approximately 1 month after the initiation of therapy, and approximately 1 month after discontinuation. The following levels are displayed: (A) 1α,25(OH)2D3, (B) serum ionized calcium, (C) 24-hour urine calcium, and (D) serum parathyroid hormone.

Both probands adhered to low calcium and oxalate diets with no excess sun exposure and copious fluid intake. Multivitamins, vitamin D supplementations, and vitamin D fortified food had been largely eliminated in both patients.

CYP24A1 activity in patient 1 and patient 2. CYP24A1 activity was measured in fibroblasts from patients 1 and 2 and compared with values for normal non-stone formers. (A) CYP24A1 activity was measured in fibroblasts from patients 1 and 2, and compared with that of normal non-stone formers (all fibroblasts are the same passage number 3). Fibroblasts from patient 1 (P1) and patient 2 (P2) produced no metabolites of 1α,25(OH)2D3. In contrast, normal fibroblasts produced metabolites that have the same retention time (18.4 minutes) of metabolites using recombinant CYP24A1 (as indicated by the arrow). Our previous study revealed that this peak contained 1-α,24R,25(OH)2D3 and 1-α,23S,25-(OH)2D3 in the ratio of 4:1 (19). Although we found no other metabolites in normal fibroblasts, further metabolites may be observed with increasing reaction time. The shoulder on the main substrate peak is the 6-s-cis form of 1- α,25(OH)2D3 generated by rotation around the 6,7 carbon bond. The interconversion between the 6-s-trans- and 6-s-cis-forms has a low energy barrier and therefore occurs rapidly in solution at room temperature. (B) CYP24A1 protein amount is reduced in patient 1 and patient 2. Western blot of whole fibroblast lysates probed with CYP24A1 antibody shows reduced protein amount in patients compared with normal. (C) β-actin serves as loading control quantification by densitometry.

Molecular Analyses

We focused our molecular analysis on candidate genes involved in vitamin D metabolism. CYP27B1 sequence analysis revealed no mutations in either patient, eliminating the possibility of constitutively upregulated production of 1α,25(OH)2D3.

However, sequencing of CYP24A1 in patient 1 revealed a heterozygous 3-bp deletion (c.428_430del; p.E143del) inherited from his mother (I-1.2) and predicted to remove a highly conserved glutamate at position 143 (Figure 4; I-2.2). Patient 1 also had a heterozygous transition of T to C (c.443T>C), inherited from his father (I-1.1) and altering a moderately conserved amino acid (p.L148P). His unaffected twin (I-2.1) harbored only the p.E143del mutation. Patient 2 exhibited bi-allelic mutations in CYP24A1, including the p.E143del deletion that he inherited from his father (Figure 4; II-1.1). The second mutation was a heterozygous transition in exon 9 (c.1226T>C), changing a moderately conserved amino acid (p.L409S); his mother (II-1.2) and brother (II-2.1) were heterozygous for this variant.

Family pedigree and molecular analysis. The pedigrees of the two families are illustrated. Affected members are shown in black squares, whereas unaffected members are denoted in white solid objects. Patient 1 (I-2.2) harbored a 3-bp deletion, p.E143del, inherited from his mother (I-1.2), and a p.L148P missense mutation that was inherited from his father (I-1.1). Patient 2 (II-2.2) also had the same p.E143del inherited from his father (II-1.1), and a p.L409S inherited from his mother (II-1.2).

Frequency of CYP24A1 Mutations

A search of dbSNP for CYP24A1 variants identified 37 nonsynonymous single nucleotide polymorphisms (SNPs). Reported MAFs within this group varied between 0.001 and 0.075 (19 SNPs); 16 SNPs had not reported MAFs, (Table 3 and Supplemental Table). We applied six pathogenicity prediction programs (Blossum 62, POLYPHEN, SIFT, Pmut, Panther, and SNAP) to estimate the deleteriousness of each variant (Supplemental Table 1).

Thirteen of the SNPs were considered likely deleterious (i.e., evaluated by POLYPHEN to have an 80%–90% chance of causing a functional defect in enzyme activity)s (Table 3). The cumulative allele frequency of these variants was 0.140. If the variant with unusually high MAF, p.M374T, was discarded, then the cumulative frequency was 0.065. These estimates do not include the 16 variants in dbSNP considered neutral or possibly deleterious by POLYPHEN, or those listed without frequencies (Supplemental Table 1). Published disease-causing mutations were also included in Supplemental Table 1; they were not reported in dbSNP as of March 2012.

Discussion

Nephrolithiasis represents a major global health problem with a lifetime prevalence estimated at 10%–15%, depending upon age, sex, race, and geographic location (21–23). Family history of stones has been reported in close to 40% of patients with nephrolithiasis (24,25). In contrast, the frequency of nephrocalcinosis is not available because most patients are asymptomatic, but detection of nephrocalcinosis is increasing due to the performance of routine diagnostic renal ultrasonography (26,27). Prevention of nephrocalcinosis and nephrolithiasis could have widespread beneficial effects, but prophylaxis should be based on understanding the etiologies.

Both nephrocalcinosis and nephrolithiasis exhibit hypercalciuria as a manifestation of abnormal calcium handling, which can be related to the level of 1α,25(OH)2D3, or calcitriol. This hormone stimulates the synthesis of epithelial calcium channels, calbindin 9, and the calcium adenosine triphosphatase pump to upregulate calcium absorption by the kidney and intestine. Elevated 1α,25(OH)2D3 can also adversely affect the kidney, because calcium ions alter mitochondrial structure and metabolism, causing damage to renal epithelial cells, tubular necrosis, and calcium deposition (10). In the intestine, excess 1α,25(OH)2D3 leads to hyperabsorptive hypercalcemia and hypercalciuria.

Because vitamin D 24-hydroxylase is the key regulator in preventing the development of high levels of 1α,25(OH)2D3 (28), we pursued a defect in this enzyme as the cause of nephrocalcinosis and nephrolithiasis in our patients. Several lines of evidence supported this hypothesis. First, both of our patients had increased 1α,25(OH)2D3 and very low levels of 24,25-(OH) 2D in the blood (Table 2). Low PTH detected in our patients is not commonly seen in patients with high urine calcium and high serum calcium. Second, the cultured fibroblasts of both patients showed diminished 1,25(OH)2D-24-hydroxylase activity (Figure 3A) and reduced amounts of Cyp24A1 protein (Figure 3, B and C). The occurrence of small concentrations of 24,25(OH)2D in serum could be explained by the presence of another enzyme, Cyp27A1, which catalyzes multiple hydroxylation steps involving vitamin D metabolites (29). The naturally occurring 24,25(OH)2D has the 24(R) configuration (30)(Figure 1).

Finally, our patients had bi-allelic mutations in the CYP24A1 gene. Patient 1 has a p.E143del, a known deleterious change, and a second variant, p.L148P. Residue 148 directly interacts with the enzyme’s substrate, and the L148P change decreases enzyme activity by 25%–50% (31,32). Patient 2 has the p.E143del as well as the previously reported p.L409S mutation (31). p.L409S weakens the binding of 1,25-dihydroxyvitamin D to 1,25(OH)2D-24-hydroxylase (32,33). The p.E143del mutation was reported by Schlingmann et al. in patients with increased sensitivity to vitamin D supplementation (11), and the same mutation has been found in patients with nephrolithiasis (34). Our pathogenicity and frequency assessment of the nonsynonymous CYP24A1 variants reported in dbSNP predicts that bi-allelic pathogenic defects in CYP24A1 may account for a significant portion of all calcium-containing renal stone patients.

Human CYP24A1 contains 514 amino acids and possesses both 23- and 24-hydroxylating activity (Figure 1) (8). The purified CYP24A1 has an absorption spectrum characteristic of P450 enzymes (35). The 1,25(OH)2D-24-hydroxylase molecule resides within the mitochondria of renal tubular cells of normal kidney (36) and is expressed by most 1α25(OH)2D3-responsive tissues (37). After the intestine and kidney, the skin has the highest CYP24A1 expression (38). The abundance of this enzyme in tissues plays a critical role in the removal of vitamin D metabolites (39). Studies of the Cyp24a1-null mouse also support a catabolic role for CYP24A1, because the clearance of 1α25(OH)2D3 is dramatically reduced in these mice; the plasma t1/2 increased from 6 to 60 h when CYP24A1 was absent. (31,37) In addition, mutant rat Cyp24a1 had less hydroxylating activity than the wild-type enzyme (40).

Despite the fact that CYP24A1 has equivalent Km values for 25(OH)D3 and 1α,25(OH)2D3 (41), our patients had normal levels of 25(OH)D3 in the face of elevated 1α,25(OH)2D3. This could be explained by the fact that 25(OH)D3 can undergo hydroxylation by two different enzymes, CYP27B1 and CYP24A1, whereas 1α,25(OH)2D3 undergoes catabolic hydroxylation only by CYP24A1.

Besides nephrocalcinosis and nephrolithiasis, osteopenia may be a clinical manifestation of 1,25(OH)2D-24-hydroxylase deficiency. Our pediatric patient manifested osteopenia of the spine and radius, and our adult patient had osteoporosis of the spine and forearm as well as osteopenia of the femoral neck. This phenomenon may be because 1α,25(OH)2D3 stimulates osteoclastic resorption of bone (42), a finding long recognized to occur in vitamin D intoxication (43).

Our adult patient with nephrolithiasis attempted several different therapies. Thiazide diuretics and sodium cellulose phosphate apparently had beneficial effects on the hypercalciuria. Ketoconazole, an inhibitor of 25-hydroxyvitamin D-1α-hydroxylase, normalized calcium, vitamin D, and PTH levels and may be an effective treatment for patients with 24-hydroxylase deficiency (12). However, additional studies are needed to assess the efficacy and safety of this regimen (44) because ketoconazole also inhibits other P450 enzymes, including the steroidogenic pathways producing testosterone, cortisol, and aldosterone (45). Extended use of ketoconazole in 1,25(OH)2D-24-hydroxylase deficiency may be problematic, although its long-term safety and efficacy in Cushing’s syndrome (up to 83 months) appears encouraging (46,47).

Previous reports have shown CYP24A1 mutations in children whose high vitamin D intake led to idiopathic infantile hypercalcemia (IIH) (11,48), and others hypothesized that CYP24A1 dysregulation causes hypercalcemia and nephrolithiasis (31). We have shown that CYP24A1 mutations are, in fact, associated with both nephrocalcinosis and nephrolithiasis, and have extended the phenotypic spectrum of CYP24A1 defects. Our findings are derived from investigations into patients with suspected novel metabolic disorders; we did not specifically target patients with renal disorders or disorders of vitamin D metabolism.

Genome-wide association studies have demonstrated the influence of CYP24A1 variants on vitamin D concentrations (49). In our patients, bi-allelic mutations in CYP24A1 (50) represent an autosomal recessive disorder involving enzyme deficiency. The sum of the MAFs of published mutations plus mutations reported as deleterious in dbSNP is 0.140. According to the Hardy-Weinberg principal [(p2) + (2pq) + (q2) = 1], the frequency of a recessive disorder with this allele frequency will be (0.140 (2)), or 1960 per 100,000 individuals in the general population. This includes the p.M374T mutation with an MAF of 0.075 (Table 3). If this most common variant is disregarded, the frequency of 1,25(OH)2D-24-hydroxylase deficiency is estimated at 420 per 100,000. The lifetime risk of renal stones in the general population is 10%, or 10,000 per 100,000, so the estimated frequency of kidney stones due to 1,25(OH)2D-24-hydroxylase deficiency will be between 420 and 1960 per 10,000, or 4%–20%. This may be an overestimate; however, some CYP24A1 polymorphisms may be associated with mild and more severe diseases, including IIH (31). Nevertheless, recognition of 1,25(OH)2D-24-hydroxylase has important implications (51), particularly in patients who have high urine calcium, high serum calcium, and low PTH, a pattern that is not commonly seen by experienced clinicians. Further studies involving a larger number of kidney stone formers are needed to determine if the hydroxylated forms of vitamin D (not 25(OH)D3 levels) should be routinely measured in patients with nephrocalcinosis, nephrolithiasis, and hypercalciuria of undetermined etiology.

In summary, we demonstrate that one cause of nephrocalcinosis and nephrolithiasis is elevated vitamin D due to 1,25(OH)2D-24-hydroxylase deficiency. Our analysis of whole exome sequencing data suggests that between 4% and 20% of all calcium-containing kidney stone patients may have this enzyme deficiency. Identification of patients with vitamin 1,25(OH)2D-24-hydroxylase deficiency could prompt salutary avoidance of vitamin D–supplemented dietary products. Our study was limited by having clinical and molecular data on only two patients, but the findings provide a basis for future investigations into the mechanism of nephrolithiasis related to vitamin D metabolism.

Disclosures

None.

Acknowledgments

This work was supported by the Intramural Research Program, National Human Genome Research Institute, National Institute of Dental and Craniofacial Diseases, and Undiagnosed Diseases Program, and Office of Rare Disorders Research, National Institutes of Health, Bethesda, Maryland.

Footnotes

Published online ahead of print. Publication date available at www.cjasn.org.

: Deficient mineralization of intramembranous bone in vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D.Endocrinology141: 2658–2666, 2000pmid:10875271